Project Summary/Abstract Human pluripotent stem cells (hPSCs) originate from either embryonic or transcription factor-induced origins. Under controlled conditions, they recapitulate cardiac development in vitro and give rise to cardiac myocytes of all subtypes. The de novo cardiac myocytes are the best surrogate for human cardiomyocytes for drug discovery, cardiotoxicity screen and cardiac cell replacement therapies. However, present technology for deriving cardiac myocytes from hPSCs has major hurdles to clear toward the stated translational goals. One major problem is heterogeneity; in a given batch, the derived cardiac myocytes are an indiscriminate blend of all major subtypes of cardiomyocytes, i.e., nodal, atrial and ventricular. No cell therapy, toxicity screen and drug discovery can afford to have undefined sub-populations of cardiac myocytes in a dish. We propose to solve this problem by imposing one of the most potent signals during cardiac development on cardiac myocyte patterning during hPSC differentiation. We hypothesize that retinoic acid (RA) signaling steers hPSCs to differentiate toward discrete populations of ventricular or atrial cardiomyocytes of the first heart field. Our preliminary data indicate that temporal and dose dependent manipulation of RA signaling leads to enriched populations of atrial or ventricular cardiomyocytes from hESCs and hiPSCs. Furthermore, the cardiac chamber-specific cardiomyocytes are functionally distinguishable in their electrical excitation and mechanical contraction. We will build on our initial observations to discover a robust and generalizable signaling axis to attain cardiac chamber-specific myocytes with genotypic and phenotypic hallmarks of the native ventricular and atrial myocytes. Successful completion of this proposal will lead to understanding of female vs. male differences in cardiogenic potential of hiPS cells and the fidelity of cardiac chamber-specific disease modeling.